EA000789B1 - PROCESS FOR THE 11beta-21-DIHYDROXY-2'-METHYL-5'beta-PREGNA-1,4-DIENO[17,16-D]OXAZOLE-3,20-DIONE - Google Patents

Abstract

1. Process for the purification of the compound llβ-21- dihydroxy-2'-methyl-5'βH-pregna-1,4-dieno[17,16-d-]-oxazoline-3,20-dione of formula I: which comprises: a) adsorbing said compound, contained into an aqueous solution resulting from fermentation broths or process streams, on an adsorbent polymeric resin having a styrenic or acrylic matrix, b) washing the resin with water, c) desorbing the said compound by eluting the resin with a suitable mixture of water with a water-miscible organic solvent, d) concentrating the eluate and recovering the compound of formula I by filtration. 2. Process according to claim 1, wherein the fermentation broth containing the compound of formula I is that obtained from the fermentation process for obtaining said compound, which comprises contacting 2'-methyl-4 pregnen-21-ol-(17a,16a-d-)oxazolinyl-3,20-dione or 2'-methyl-4-pregnen-21-acetyloxy-(17a,16a-d-) oxazolinyl-3,20-dione with a sequentially growing mixed culture of a Curvularia strain and an Arthrobacter strain. 3. Process according to claim 1 or 2 wherein the eluting miture of step c) contains about 30% 4. Process according to claim 2 or 3 wherein: a) the fermentation mass is first filtered, the mycelial cake is repeatedly washed with water and the washings are then combined with the filtered broth; b) the filtered broth combined with the washings is then applied on the top of a column containing the aqueousswollen adsorbing resin; b') the resin is washed with water; c) the compound of formula I is desorbed from the resin by eluting with a mixture 30/70 water/acetone; d) the eluate is concentrated and the compound of formula I is recovered by filtration. 5. Process according to claim 4 wherein the purification procedure is repeated on the combined mother liquors and the washings obtained from the elution according to steps b) and b'). 6. Process according to claim 1, 2, 3, 4 or 5 wherein the adsorbent polymeric resin is a polymeric resin having a styrenic or acrylic matrix having a particle size of about 20-50 mesh and with the following average physical characteristics: Porosity volume: about 30%-75%; Surface area: about 140-800 m<2>/g; Skeletal density: about 1.06-1.10 g/ml for styrenic resins, about 1.20-1.26 g/ml for acrylic resins; Average pore diameter: about 20-100 angstrom . 7. Process according to claim 1, 2, 3, 4 or 5 wherein the adsorbent polymeric resin is an acrylic based polymeric resins having a particle size of about 20-50 mesh and the following average physical characteristics: Porosity volume: about 30%-60%; Surface area: about 350-550 m<2>/g; Skeletal density: about 1.24-1.25 g/ml; Average pore diameter: about 20-80 angstrom . 8. Process according to claim 1, 2, 3, 4 or 5 wherein the adsorbent polymeric resin is an acrylic based polymeric resins having a particle size of about 20-50 mesh and the following average physical characteristics: Porosity volume: about 30%-60%; Surface area: about 350-550 m<2>/g; Skeletal density: about 1.25 g/ml; Average pore diameter: about 20-40 angstrom . 9. Process according to claim 1, 2, 3, 4, 6 or 7 wherein the water miscible organic solvent is a lower ketone or a lower alcohol. 10. Process according to claim 1, 2, 3, 4, 6 or 7 wherein the organic solvent is acetone. 11. Process according to claim 1, 2, 3, 4, 6 or 7 wherein the eluting mixture is a mixture 30/70 water/acetone.

Description

The present invention is about a new method for purifying the compound 11β-21 dihydroxy-2'-methyl-5'bH-pregna-1,4-dieno (17, 16-di-) oxazolin-Z, 20-dione of the formula I

deflazacort (TIN is the international non-proprietary name), in which the acetate fragment of C-21 deflazacort is replaced by a hydroxyl fragment.

Deflazacort is a compound used in therapy for several years as a calcium-retaining glucocorticoid agent.

These compounds belong to a more general class of pregno-oxazolines with anti-inflammatory, glucocorticoid and hormone-like pharmacological activities. Examples of compounds of the above class are shown in US patent No. 3413286 and No. 4440764.

A method for producing a compound of formula I is described in European patent EP-B-322630, where the aforementioned compound relates to 11 β21-dihydroxy-2'-methyl-5'bH-pregna-1,4 dieno (17,16-di-) oxazolin-3 , 20 dione.

According to the fermentation process described in the above EP-B-322630, 2'-methyl4-pregnen-21-ol- (17a, 16a-di-) oxazolinyl-3,20dion or 2'-methyl-4-pregnen-21-acetyloxy (17a, 16a-di-) oxazolinyl-3,20-dione binds to successively growing mixed cultures of Curvularia and Arthrobacter strains. In more detail, according to the invention, the aforementioned compound is preferably added to a growing culture of C. lunata NRRL 2380 in an appropriate fermentation medium 1 to 24 hours after sowing, and an 18-36 hour growing culture A is added to this mixture 48 to 72 hours after sowing. simplex ATCC 6946 and further cultivated for 40-55 hours; further fermentation is carried out under the following conditions: the temperature is maintained between 27 and 32 ° C and the pH is between 6 and 8; the fermentation product of formula I is then recovered by extracting the fermentation broth with an organic solvent (for example, chloroform), the organic extracts are concentrated and a non-solvent is added in which the compound precipitates (for example, petroleum ether).

Due to the fact that the concentration of the compound of formula I in the fermentation broth is very low, a large amount of organic solvent is necessary for the complete extraction of the compound. The use of large quantities of organic solvents, in particular halogenated solvents, can cause some problems related to labor protection and environmental protection. It has now been found that a compound of formula I can be recovered from an aqueous solution obtained from fermentation broths or from streams during the process by adsorption of a compound contained in the aforementioned aqueous solution onto an adsorption polymer resin having styrene or acrylic matrices, followed by desorption of the aforementioned compound by eluting the resin with a suitable mixture of water with water-miscible organic solvents.

Typical aqueous solutions containing a compound of formula I and accompanied by undesirable accompanying products are filtrates of fermentation broths from the corresponding mycelia, optionally together with aqueous washings of the aforementioned mycelia, or partially purified streams during the process. Examples of undesirable accompanying products are colored impurities, by-products, unspent starting materials, salts and water-soluble components of the fermentation medium.

In particular, the purification process according to the invention can be successfully applied to obtain a compound of formula I from a filtration of a fermentation broth resulting from the fermentation process described in European patent B-322630.

Suitable resins for the cleaning process can have, on average, particles of about 20-50 mesh in size and the following average physical characteristics:

Free volume Surface area Skeleton density about 30-75% about 140-800 m ² / g about 1.06-1.10 g / ml (styrene resins) about 1.20-1, 26 g / ml (acrylic resins)

The average pore diameter of about 20-100 A

Examples of styrene polymer resin adsorbents suitable for the aforementioned extraction process are commercially available resins such as Kastel® S / 112 (Dow Chemical), Amberlite® XAD / 2, XAD / 4 or XAD / 16 (Rohm & Haas), or the like. Examples of adsorbents based on acrylic polymer resins suitable for the aforementioned extraction process are commercially available resins such as Kastel® S / 221 or S / 223 (Dow Chemical), Amberlite® XAD / 7 or XAD / 8 (Rohm & Haas) or like them.

In general, for the process according to the invention, the use of resins based on acrylic polymers is preferable, especially for resins with a particle size of about 20-50 mesh, which have the following average physical characteristics:

Free volume Surface area Skeleton density Average pore diameter of about 30-60%, about 350-550 m ² / g; about 1.24-1.25 g / ml about 20-80 A

Examples of adsorbents for acrylic-based polymer resins with the aforementioned characteristics that may be suitable for use are mentioned previously by Kastel® S / 221 and Amberlite® XAD / 7.

Particularly preferred for the present cleaning process are acrylic-based polymeric resins having particles of about 20-50 mesh in size and the following average physical characteristics:

Free volume Surface area Skeleton density Average pore diameter about 30-60% about 350-550 m ² / g about 1.25 g / ml about 20-40 A

As an example, a commercially available Kastel® S / 221 resin is suitable for use.

Suitable for elution are mixtures containing from 0 to 80% water with water-miscible organic solvents, such as lower ketones (eg, acetone, ethyl methyl ketone); lower alcohols (e.g. methanol, ethanol, propanol, butanol); and the like.

Mixtures containing from 10 to 50% water with one of the aforementioned organic solvents are preferred, a mixture containing about 30% water is particularly preferred. Acetone is the preferred solvent.

When using the process according to the invention for the purification of a compound of formula I obtained according to the fermentation process described in patent EP-B-322630, the following basic procedures are used.

When the aforementioned fermentation process is completed, the fermentation mass is firstly filtered by a known technique. The mycelium filter cake is washed repeatedly with water, and the washings are then combined with the broth filtrate. Alternatively, the mycelium may be washed with an organic solvent selected from those previously listed in order to recover the activity contained therein; in this case, the washings are combined with the broth filtrate only after removal of the solvent, for example, by distillation under vacuum.

The broth filtrate is combined with washings and then applied to a column containing an adsorption resin; usually the eluted solution contains only traces of the product. The resin is then washed with water to remove salts and other water-soluble impurities (like the above, this wash also mainly contains only traces of the product).

The compound of formula I is then stripped from the resin by elution with a mixture of water and an organic solvent, as defined above, preferably a mixture of water and acetone in a ratio of 30/70. The eluate was concentrated and the product was recovered by filtration. In this procedure, the bulk of the product is obtained from the broth filtrate; it is shown that, as a rule, more than 90% of the total amount of the desired compound, initially placed on the column, is recovered during the first elution.

Residual amounts can be recovered by repeating the above procedure, after combining the mother liquor from the first elution and washings with water. The total yield is about 96%.

To best illustrate the invention, the following examples are given.

Example 1. Sequential growth of C. lunata and A. simplex.

I) Nutrient medium for mowed agar

Wednesday Saburo (for S. lunata)

Antibiotic Agar No. 1 (for A. simplex)

II) Vegetative and precultural environment

a) for C. lunata

Soya flour 1 3 g / l KH ₂ PO ₄ 5 g / l Dextrose 10 g / l Peptone 5 g / l Before autoclaving establish pH 6.5-7.5. b) for A. simplex Dextrose 1.0 g / l Soya flour 5.0 g / l Peptone 5.0 g / l Basamin busch 3.0 g / l KH2PO4 5.0 g / l NaCl 5.0 g / l Silicone 0.1 ml / l Before autoclaving establish

pH 6.5-7.5.

Sh) Fermentation medium

The fermentation medium has the same composition as the pre-culture medium for Olunata, as reported above.

1U) Fermentation technique

Mowed agar was used in separate inoculated 500 ml vials, which were cultured at a temperature of about 28 ° C for 12-24 hours (C. lunata) or 18-36 hours (A. simplex) in the presence of 10 ml of the vegetative medium indicated above. This seed was used in the method described below.

Aliquots (from 1 to 5%) of C. lunata culture, obtained as described above, were transferred to an 8 liter fermenter containing the fermentation medium reported above and cultured for about 24 hours at 29-32 ° C.

Then, 4 g of 2'-methyl-4-pregnen-21ol- (17a, 16a-di) -oxazolinyl-3,20-dione were added and fermentation was continued for about 36-72 hours, counting from seeding.

Then, an A. simplex culture of 18-36 hours was added to the fermenter, and fermentation was continued for the next 40-55 hours.

The reaction was monitored using a known TLC method or, more preferably, HPLC, observing the disappearance of the starting material and / or the appearance of the final product. As an additional control, the appearance or disappearance of intermediates can be monitored. The yield of the converted product, determined by HPLC, was 70-75%.

Example 2. Obtaining the compounds of formula I.

40-55 hours after the addition of A. simplex, the conversion of the substance can generally be considered complete, and the fermentation mass can be used to isolate the desired substance of formula I.

The pH of the fermentation mass was adjusted to approximately 3-4 with H ₂ SO ₄ , this mixture was filtered using filtration devices through filtered mycelium, then washed repeatedly with acidified water (3 <pH <4). Filtered broth and washings were combined to obtain 11 L of a mixture containing a compound of formula I having a characteristic of 270 ppm (obtained by HPLC on silica gel; Spherisorb 3μιη column, 100 x 4.6 mm; flow rate 1.3 ml / min, the mobile phase is 0.0025 M NaH ₂ PO ₄ : CH ₃ CN 7: 3, UV registration at a wavelength of 254 nm). The mixture was then applied at room temperature to the top of a chromatographic column (6 x 11 cm) containing about 300 ml of water-swollen acrylic-based polymer resin (Kastel ^® S / 221, Dow Chemical), at a flow rate of about 300 ml / h . Unbound column material (stock solutions) that contained less than 2 ppm of activity was collected separately.

The resin was then washed with 600 ml of deionized water to remove salts and other water-soluble impurities. Again, swabs containing less than 2 ppm of activity were collected separately.

The resin bound to the column was then stripped, eluting with a 70/30 mixture of acetone and water at a rate of about 1 50 ml / h, collecting about 200 ml of eluate.

The eluate was concentrated in vacuo to approximately 10 ml, while the product was precipitated.

The suspension was cooled to approximately 5 ° C and after 2 hours the precipitate was separated by filtration and washed with cold water. After drying under vacuum at 50 ° C, 2.8 g of the compound of formula I were obtained (purity 98%).

The mother liquors and washings were combined and, after dilution with water (about 1-2 volumes of water relative to 1 volume of the mother liquor), were applied by direct current to a column (2 x 1 3 cm) containing 40 ml of the above swollen resin at a rate of about 40 ml / hours

After the resin was washed with 80 ml of water, the product bound on the column was desorbed by elution using a mixture of acetone and water in a ratio of 70/30 at a rate of 20 ml / h. After concentration in vacuo, cooling, filtration, washing with cold water and drying, the following 0.1 4 g of the compound of formula I were obtained.

The total yield of the substance from the fermentation mass was usually about 96%.

When repeating the methods described in examples 1 and 2, but when using 2'-methyl4-pregnen-21-acetyloxy- (1 7a, 1 6a-di) -oxazolinyl-3,20-dione instead of 2'-methyl-4- pregnen-21ol- (1 7a, 1 6a-di) -oxazolinyl-3, 20-dione, the compound of formula I was obtained in practically the same yield.

Claims (10)

CLAIM 1. The method of purification of the compound 11β-21dihydroxy-2'-methyl-5'v H-pregna-1,4-dieno [17, 16-di-] -oxazolin-3,20-dione of the formula, which includes: a) adsorption of the above-named compound contained in an aqueous solution obtained from fermentation broths or from streams during the process on an adsorbing polymer resin based on styrene or acrylic matrices, b) washing the resin with water c) desorbing a compound of formula I from a resin by elution with a mixture containing 1050% water and a water-miscible organic solvent, g) the concentration of the eluate and the extraction from it of the compounds of formula I by filtration. 2. The method according to claim 1, wherein the fermentation broth containing the compound of formula I was obtained as a result of a fermentation process to produce the title compound, which, in turn, involves the interaction of the 2'-methyl-4-pregnen-21-one (17a, 16adi-) oxazolinyl-3,20-dione or 2'-methyl-4pregnen-21-acetyloxy- (1 7a, 1 6a-di) oxazolinyl-3,20-dione with successively growing mixed cultures of the Curvularia strains and Arthrobacter. 3. The method according to claim 1 or 2, in which the eluting mixture of step c) contains about 30% water. 4. The method according to claims 1 and 2, in which a) the fermentation mass is first filtered, the mycelial precipitate on the filter is repeatedly washed with water, and the washings are combined with the broth filtrate; b) the filtered broth combined with the washout is then applied to the top of a column containing an adsorption resin swollen with water; b ') the resin is washed with water; c) a compound of formula I is desorbed from the resin by elution with a mixture of water and acetone in a ratio of 30/70; g) the eluate is concentrated and the compound of formula I is recovered by filtration. 5. The method according to claim 4, in which the cleaning process is repeated on the combined fraction of mother liquors and washes, obtained by elution in steps b) and b '). 6. The method according to claims 1, 2, 3, 4 or 5, in which a polymeric resin based on styrene or acrylic matrices with a particle size of 20-50 mesh, having the following averaged physical characteristics is used as the adsorption polymer resin, having the following averaged physical characteristics: Free volume about 30 -75% Surface area about 140-800 m ² / g Skeleton density for styrene resins about 1.06-1.10 g / ml for acrylic resins about 1.2-1.26 g / ml The average pore diameter of about 20-100 A 7. The method according to claims 1, 2, 3, 4 or 5, in which as the adsorption polymer resin is used acrylic-based polymer resin having particles of about 20-50 mesh in size and the following averaged physical characteristics: Free volume about 30-60% Surface area about 350-550 m ² / g Skeleton density of about 1.24-1, 25 g / ml The average pore diameter of about 20-80 A 8. The method according to claims 1, 2, 3, 4 or 5, in which as the adsorption polymer resin is used acrylic-based polymer resin having particles of about 20-50 mesh in size and the following averaged physical characteristics: Free volume about Surface area about Skeleton density about Average pore diameter about 9. The method according to claims 1, 2, 3, 4, rum water-miscible organic solvent is a lower ketone or a lower alcohol. 10. The method according to claims 1, 2, 3, 4, 6 or 7, in which the organic solvent is acetone. 30-60% 350-550 m ² / g 1.25 g / ml 20-40 A 6 or 7, in which 11. A method according to claims 1, 2, 3, 6 or 7, in which the eluting mixture is a mixture of water and acetone in a ratio of 30/70.